Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells and lymphocytes. During the past year we completed an ongoing clinical trial of retroviral gene transfer into CD34+ cells from patients with Gaucher disease, showing engraftment with corrected cells at low levels, even without prior myeloablation. In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on the issue of ex vivo expansion after retroviral transduction. Prolonged ex vivo expansion has been shown to profoundly engraftment of genetically-marked cells, despite marked expansion of cells assayable in vitro, such as CFU-GM and total CD34+ cells. This finding has important clinical ramifications in design of both gene therapy and standard transplantation trials. We have been encouraged to discover that the inclusion of the cytokine flt 3 ligand and either autologous stromal cells or fibronectin improves gene transfer efficiency into engrafting cells to levels of 10-50%, a range with clinical utility. These high levels have also allowed for the first time in a large animal model assessment of contribution of specific marked clones to different lineages over time, using inverse PCR and insertion site analysis. Multiple clones contribute at least for the first 1-4 months analyzed to date. Other ongoing projects in the lab involve the assessment of the effect of foreign expressed genes in vector constructs on levels of gene-modified cells in vivo, using either CD34+ primitive cells or lymphocytes as targets. Finally, rhesus lymphocytes have been targets for head-to-head comparison of adeno-associated virus and retroviral vectors. Both AAV and retrovirus produce excellent gene transfer levels in vivo post-infusion, but AAV-marked cells disappear after 5-6 weeks, due to only episomal and not integrated forms of the vector.